Microglia proliferation is regulated by hydrogen peroxide from NADPH oxidase

Microglia are resident brain macrophages that become activated and proliferate following brain damage or stimulation by immune mediators, such as IL-1beta or TNF-alpha. We investigated the mechanisms by which microglial proliferation is regulated in primary cultures of rat glia. We found that basal proliferation of microglia was stimulated by proinflammatory cytokines IL-1beta or TNF-alpha, and this proliferation was completely inhibited by catalase, implicating hydrogen peroxide as a mediator of proliferation. In addition, inhibitors of NADPH oxidase (diphenylene iodonium or apocynin) also prevented microglia proliferation, suggesting that this may be the source of hydrogen peroxide. IL-1beta and TNF-alpha rapidly stimulated the rate of hydrogen peroxide produced by isolated microglia, and this was inhibited by diphenylene iodonium, implying that the cytokines were acting directly on microglia to stimulate the NADPH oxidase. Low concentrations of PMA or arachidonic acid (known activators of NADPH oxidase) or xanthine/xanthine oxidase or glucose oxidase (generating hydrogen peroxide) also increased microglia proliferation and this was blocked by catalase, showing that NADPH oxidase activation or hydrogen peroxide was sufficient to stimulate microglia proliferation. In contrast to microglia, the proliferation of astrocytes was unaffected by the presence of catalase. In conclusion, these findings indicate that microglial proliferation in response to IL-1beta or TNF-alpha is mediated by hydrogen peroxide from NADPH oxidase.     

Gibberellin structure and florigenic activity in Lolium temulentum,a long-day plant

Structural requirements for florigenic activity among gibberellins (GAs) and GA derivatives, including several new ones, applied once to leaves of Lolium temulentum, were examined. The compounds were applied to plants kept either in non-inductive short days (SD) or exposed to one inductive long day (LD). Inflorescence initiation and stem-elongation responses were assessed three weeks later. Among the GAs used, the range in effective dose for inflorescence initiation was more than 1000-fold, but substantially less for stem elongation. Some GAs promoted both stem elongation and inflorescence initiation, some promoted one without the other, and some affected neither. The structural features enhancing florigenic activity were often different from those enhancing stem elongation. Except in the case of 2,2-dimethyl GA₄, a double bond in the A ring at either C-1,2 or C-2,3 was essential for high florigenic activity, though not for stem elongation. A free carboxy group was needed for both. Inflorescence initiation in Lolium was enhanced by hydroxylation at C-12, ?13 and ?15, whereas hydroxylation at C-3 reduced the effect on inflorescence initiation but increased that on stem elongation. A 12β-hydroxyl was more effective than the α epimer for inflorescence initiation whereas the reverse was true for stem elongation. Although such differential effectiveness of GAs for inflorescence initiation and for stem elongation could reflect differences in uptake, transport or metabolism, we suggest that it is indicative of specific structural requirements for inflorescence initiation.     

Sequence analysis of sub-genotype D hepatitis B surface antigens isolated from Jeddah,Saudi Arabia

Little is known about the prevalence of HBV genotypes/sub-genotypes in Jeddah province, although the hepatitis B virus (HBV) was identified as the most predominant type of hepatitis in Saudi Arabia. To characterize HBV genotypes/sub-genotypes, serum samples from 15 patients with chronic HBV were collected and subjected to HBsAg gene amplification and sequence analysis. Phylogenetic analysis of the HBsAg gene sequences revealed that 11 (48%) isolates belonged to HBV/D while 4 (18%) were associated with HBV/C. Notably, a HBV/D sub-genotype phylogenetic tree identified that eight current isolates (72%) belonged to HBV/D1, whereas three isolates (28%) appeared to be more closely related to HBV/D5, although they formed a novel cluster supported by a branch with 99% bootstrap value. Isolates belonging to D1 were grouped in one branch and seemed to be more closely related to various strains isolated from different countries. For further determination of whether the three current isolates belonged to HBV/D5 or represented a novel sub-genotype, HBV/DA, whole HBV genome sequences would be required. In the present study, we verified that HBV/D1 is the most prevalent HBV sub-genotype in Jeddah, and identified novel variant mutations suggesting that an additional sub-genotype designated HBV/DA should be proposed. Overall, the results of the present HBsAg sequence analyses provide us with insights regarding the nucleotide differences between the present HBsAg/D isolates identified in the populace of Jeddah, Saudi Arabia and those previously isolated worldwide. Additional studies with large numbers of subjects in other areas might lead to the discovery of the specific HBV strain genotypes or even additional new sub-genotypes that are circulating in Saudi Arabia.     

Long-term effects on the nitrogen budget of a short-rotation grey alder (Alnus incana (L.) Moench) forest on abandoned agricultural land

Short-rotation energy forestry is one of the potential ways for management of abandoned agricultural areas. It helps sequestrate carbon and mitigate human-induced climate changes. Owing to symbiotic dinitrogen (N₂) fixation by actinomycetes and the soil fertilizing capacity and fast biomass growth of grey alders, the latter can be suitable species for short-rotation forestry. In our study of a young grey alder stand (Alnus incana (L.) Moench) on abandoned arable land in Estonia we tested the following hypotheses: (1) afforestation of abandoned agricultural land by grey alder significantly affects the soil nitrogen (N) status already during the first rotation period; (2) input of symbiotic fixation covers an essential part of the plant annual N demand of the stand; (3) despite a considerable N input into the ecosystem of a young alder stand, there will occur no significant environmental hazards (N leaching or N₂O emissions). The first two hypotheses can be accepted: there was a significant increase in N and C content in the topsoil (from 0.11 to 0.14%, and from 1.4 to 1.7%, respectively), and N fixation (151.5 kg N ha⁻¹ yr⁻¹) covered about 74% of the annual N demand of the stand. The third hypothesis met support as well: N₂O emissions (0.5 kg N ha⁻¹ yr⁻¹) were low, while most of the annual gaseous N losses were in the form of N₂ (73.8 kg N ha⁻¹ yr⁻¹). Annual average NO₃-N leaching was 15 kg N ha⁻¹ yr⁻¹ but the N that leached from topsoil accumulated in deeper soil layers. The soil acidifying effect of alders was clearly evident; during the 14-year period soil acidity increased 1.3 units in the upper 0-10 cm topsoil layer.     

Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.     

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.     

Increased organic carbon concentrations in Estonian rivers in the period 1992–2007 as affected by deepening droughts

There are 10,091 km² of peatlands in Estonia and human activities may have changed the role of northern peatlands from global sinks to global sources of carbon. The aim of this work was to explain the changes in organic carbon exports from eleven Estonian rivers in the period 1992–2007 in terms of land use change, climate change quantified by trends in stream-water discharge and hydrological droughts and reductions in atmospheric sulfate quantified by change in water chemistry. Direct TOC (total organic carbon) measurements had been initiated in 1998. We used CODKMnO₄ (permanganate oxygen consumed) as its surrogate for the whole time span (Spearman’s determination coefficient in six small northern Estonian rivers 0.95 > ρ² > 0.72; p < 0.01). The Mann–Kendall test revealed significant trends in the TOC proxy in five small rivers in northern Estonia (M–K stat > 2.35; p < 0.05). The trends in the eleven investigated streams correlated closely with the increased 28 ratios of drought days (Spearman’s ρ² = 0.68; p < 0.01). The correlations with land use compositions, decreases in water discharges and SO₄ ^2? concentrations were insignificant (p > 0.05). We conclude that the main factor in the increase of organic carbon export is the deepening of droughts driven by climate change, magnified by man-made drainage.